NEUTRALIZING HUMAN MONOCLONAL-ANTIBODIES AGAINST PUUMALA VIRUS, CAUSATIVE AGENT OF NEPHROPATHIA-EPIDEMICA - A NOVEL METHOD USING ANTIGEN-COATED MAGNETIC BEADS FOR SPECIFIC B-CELL ISOLATION

Human monoclonal antibodies (MAbs) against Puumala (PUU) virus were ge nerated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein -Barr virus transformation. Four IgG-positive monoclonal lymphoblastoi d cell lines (LCLs) were established and have remained stable MAb secr etors for over 12 months. Analyses of the antigen and epitope specific ities recognized by the MAbs showed overlapping binding patterns of fo ur antiglycoprotein 2-specific clones. Identical isotypes (IgG1 lambda ) and isoelectric points (9-2) of the four MAbs suggested that they we re derived from the same original clone. The MAbs reacted with eight P UU virus-like strains, but were negative for Hantaan, Seoul, and Prosp ect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European f orm of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a foc us reduction neutralization test. The MAb neutralizing activity was si gnificantly enhanced in the presence of human or guinea-pig complement . To stabilize and increase antibody secretion and to reduce the deman d for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human x mouse partn er SPAM-8. Several of the established human x (human x mouse) monoclon al triomas grew faster and produced larger amounts of MAbs when compar ed with the original LCLs.